Abstract
This study aimed to investigate the effects of cyclopamine, a specific inhibitor of Hedgehog signaling pathways, on the chondrogenic differentiation of mesenchymal stem cells (MSCs). During culture, the experimental groups were treated with cyclopamine and their cell proliferation status was assessed using the MTT test. The extra-bone cellular matrix (ECM) and Collagen II (Col II) was detected by toluidine blue staining and immunohistochemistry of cells. The concentrations of Col II and aggrecan in the culture solution and cytosol were detected using ELISA on the 7th, 14th, and 21st days of cyclopamine induction. Gene and protein expression of Col II and aggrecan were analyzed on the 14th day of cyclopamine induction using real-time PCR and western blot analyses. No significant differences in proliferation of mesenchymal stem cells were found between the control group and the group treated with cyclopamine. Compared to the blank control group, the ECM level was low and the protein and mRNA concentrations of Collagen II (Col II) and aggrecan in the culture solution and cytosol, respectively, were significantly reduced in the experimental group. The Smo acted as a key point in the regulations of Hedgehog signaling pathway on the chondrogenic differentiation of rabbit MSCs.
Highlights
Hedgehog, a protein encoded by the segment polarity gene Hedgehog, plays an important role in embryonic development and organ formation
Another factor was the influence of fetal bovine serum (FBS) on the chondrogenesis of rabbit mesenchymal stem cells (MSCs). 10% FBS has been reported to have promoting effect for MSCs chondrogenesis and been utilized as a component among chondrogenesis inducer [18].We tried to keep the uniform concentration of FBS applied to avoid the experimental bias
We deduced that the Smo molecular might play as a key factor in the signal transduction process, and Collagen II (Col II) and aggrecan were found as the target genes up-regulated by Hh during rabbit MSCs chondrogenesis
Summary
A protein encoded by the segment polarity gene Hedgehog, plays an important role in embryonic development and organ formation. Almost all processes of embryonic development are regulated by Hedgehog. Much has been learned about Hedgehog and its signal transduction pathway, concerning a novel and important function in bone and cartilage formation [1,2,3]. The Hedgehog gene family contains at least three counterparts, referred to as Shh (Sonic hedgehog), Ihh (Indian hedgehog), and Dhh (Desert hedgehog). When Hh is bound to Ptch, Smo performs its regulatory role and is activated to enhance the transcription activities of its target genes, which leads to the activation of a series of downstream signal molecules
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